Cell preamble runx3 recombinant proteins, polynucleotides encoding the same, and anticancer compositions including the same

Abstract

The present invention discloses cell permeable RUNX3 recombinant proteins where a Macromolecule Transduction Domain (MTD) is fused to a tumor and metastasis suppressor RUNX3. Also disclosed are polynucleotides encoding the cell permeable RUNX3 recombinant proteins, an expression vector containing the cell permeable RUNX3 recombinant protein, and a pharmaceutical composition for preventing metastasis which contains the cell permeable RUNX3 recombinant protein as an effective ingredient. The cell permeable RUNX3 recombinant proteins of the present invention can induce the reactivation of TGF-β signal transduction pathway which causes cell cycle arrest by efficiently introducing a tumor and metastasis suppressor RUNX3 into a cell. Therefore, the cell permeable RUNX3 recombinant proteins of the present invention can be effectively used as an anticancer agent capable of preventing cancer growth and metastasis by suppressing the proliferation, differentiation, and migration of cancer cells.

1 . A cell permeable RUNX3 recombinant protein comprising a macromolecule transduction domain (MTD) and a human tumor and metastasis suppressor RUNX3, said MTD being fused to N-terminus, C-terminus, or both termini of the human tumor and metastasis suppressor RUNX3. 
     
     
         2 . The cell permeable RUNX3 recombinant protein according to  claim 1 , wherein the human tumor and metastasis suppressor RUNX3 is in a full-length form having an amino acid sequence represented by SEQ ID NO: 2 which includes all of N-terminal, R-terminal and PST-rich domains, or a truncated form lacking one or more of the N-terminal, R-terminal and PST-rich domains. 
     
     
         3 . The cell permeable RUNX3 recombinant protein according to  claim 1 , wherein the MTD has an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 to 196. 
     
     
         4 . The cell permeable RUNX3 recombinant protein according to  claim 3 , wherein the MTD is selected from the group consisting of a kFGF4 (kaposi fibroblast growth factor 4)-derived MTD having an amino acid sequence represented by SEQ ID NO: 3, a JO-13 MTD having an amino acid sequence represented by SEQ ID NO: 16, a JO-57 MTD having an amino acid sequence represented by SEQ ID NO: 60, a JO-85 MTD having an amino acid sequence represented by SEQ ID NO: 88, and a JO-108 MTD having an amino acid sequence represented by SEQ ID NO: 111. 
     
     
         5 . The cell permeable RUNX3 recombinant protein according to  claim 1 , further comprising:
 a nuclear localization sequence (NLS) and a histidine-tag affinity domain, said nuclear localization sequence and histidine-tag affinity domain being covalently coupled to one end of the recombinant protein.   
     
     
         6 . The cell permeable RUNX3 recombinant protein according to any one of  claims 1  to  5 , wherein the recombinant protein is selected from the group consisting of:
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to the N-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to the C-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to both termini of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to the C-terminus of a RUNX3 N-terminal domain fragment lacking R-terminal and PST-rich domains in the amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to the C-terminus of a RUNX3 R-terminal domain fragment lacking N-terminal and PST-rich domains in the amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to the C-terminus of a RUNX3 PST-rich domain fragment lacking N- and R-terminal domains in the amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to the C-terminus of a RUNX3 N- and R-terminal domain fragment lacking a PST-rich domain in the amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to the C-terminus of a RUNX3 R-terminal and PST-rich domain fragment lacking an N-terminal domain in the amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a kFGF4-derived MTD having an amino acid sequence represented by SEQ ID NO: 3 is fused to the C-terminus of a portion of a RUNX3 R-terminal and PST-rich domain fragment lacking an N-terminal domain which corresponds to amino acid residues 68-200 in the amino acid sequence of SEQ ID NO: 2; 
 a recombinant protein wherein a JO-57 MTD having an amino acid sequence represented by SEQ ID NO: 60 is fused to the N-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-57 MTD having an amino acid sequence represented by SEQ ID NO: 60 is fused to the C-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-57 MTD having an amino acid sequence represented by SEQ ID NO: 60 is fused to both termini of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-85 MTD having an amino acid sequence represented by SEQ ID NO: 88 is fused to the N-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-85 MTD having an amino acid sequence represented by SEQ ID NO: 88 is fused to the C-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-85 MTD having an amino acid sequence represented by SEQ ID NO: 88 is fused to both termini of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-13 MTD having an amino acid sequence represented by SEQ ID NO: 16 is fused to the N-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-13 MTD having an amino acid sequence represented by SEQ ID NO: 16 is fused to the C-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-13 MTD having an amino acid sequence represented by SEQ ID NO: 16 is fused to both termini of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-108 MTD having an amino acid sequence represented by SEQ ID NO: 111 is fused to the N-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 a recombinant protein wherein a JO-108 MTD having an amino acid sequence represented by SEQ ID NO: 111 is fused to the C-terminus of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; and 
 a recombinant protein wherein a JO-108 MTD having an amino acid sequence represented by SEQ ID NO: 111 is fused to both termini of a full-length RUNX3 having an amino acid sequence represented by SEQ ID NO: 2; 
 wherein a His-tag and a NLS derived from SV40 large T antigen are covalently coupled to the N-terminus of the above constructs. 
 
     
     
         7 . The cell permeable RUNX3 recombinant protein according to  claim 1 , wherein the recombinant protein has an amino acid sequence selected from the group consisting of SEQ ID NOS: 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237 and 239. 
     
     
         8 . A polynucleotide encoding the cell permeable RUNX3 recombinant protein according to  claim 1 . 
     
     
         9 . The polynucleotide according to  claim 8 , wherein the polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NOS: 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 226, 228, 230, 232, 234, 236 and 238. 
     
     
         10 . An expression vector comprising the polynucleotide according to  claim 8 . 
     
     
         11 . The expression vector according to  claim 10 , wherein the expression vector is selected from the group consisting of:
 pHM 1 R3 which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 198 which encodes cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHR3M 1  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 200 which encodes a cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHM 1 R3M 1  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 202 which encodes a cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHR3NM 1  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 204 which encodes a cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHR3RM 1  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 206 which encodes a cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHR3PM 1  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 208 which encodes a cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHR3NRM 1  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 210 which encodes a cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHR3RPM 1  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 212 which encodes a cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHR3CRM 1  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 214 which encodes a cell permeable RUNX3 recombinant protein fused to a kFGF4-derived MTD;   pHM 2 R3 which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 216 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-57 MTD;   pHR3M 2  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 218 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-57 MTD;   pHM 2 R3M 2  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 220 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-57 MTD;   pHM 3 R3 which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 222 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-57 MTD;   pHR3M 3  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 224 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-85 MTD;   pHM 3 R3M 3  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 226 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-85 MTD;   pHM 4 R3 which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 228 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-13 MTD;   pHR3M 4  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 230 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-13 MTD;   pHM 4 R3M 4  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 232 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-13 MTD;   pHM 5 R3 which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 234 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-108 MTD;   pHR3M 5  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 236 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-108 MTD; and   pHM 5 R3M 5  which comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 238 which encodes a cell permeable RUNX3 recombinant protein fused to a JO-108 MTD;   
     
     
         12 . A transformant comprising the expression vector according to  claim 10 . 
     
     
         13 . The transformant according to  claim 12 , wherein the transformant is  E. coli  DH5α/HM 2 R3 (KCTC-11408BP). 
     
     
         14 . The transformant according to  claim 12 , wherein the transformant is  E. coli  DH5α/HM 3 R3 (KCTC-11409BP). 
     
     
         15 . A method of producing a cell permeable RUNX3 recombinant protein comprising the following steps of:
 1) culturing the transformant according to  claim 12  to express a cell permeable RUNX3 recombinant protein; and   2) purifying the expressed cell permeable RUNX3 recombinant protein from the culture containing the transformant.   
     
     
         16 . A pharmaceutical composition for treating RUNX3 deficiency or failure comprising the cell permeable RUNX3 recombinant protein according to  claim 1  as an effective ingredient and a pharmaceutically acceptable carrier. 
     
     
         17 . The pharmaceutical composition according to  claim 16 , which is used for preventing or treating tumor growth and metastasis, wherein the tumor is selected from the group consisting of gastric cancer, pancreatic cancer, lung cancer, colon cancer and liver cancer.